Antibiotic resistance is a looming global problem threatening some of the most significant public health gains of the past century. This critical health challenge, together with the emerging threat of biowarfare agents calls for the identification and development of new strategies for the treatment of bacterial infections. Targeting bacterial virulence systems, such as extracellular secretion pathways, is an attractive therapeutic alternative to conventional antibiotic therapy since it attenuates the pathogens without killing them possibly reducing the emergence of drug resistance. The type II secretion (T2S) system is widely distributed among gram-negative pathogens where it secretes a variety of toxins and degradative enzymes, making it an ideal target for therapeutic intervention. It consists of a multiprotein complex that spans the entire cell envelope, including an ATPase in the cytoplasm, an inner membrane subcomplex that extends into the periplasmic compartment, and a secretion pore in the outer membrane. Since several of these components are unique to TIS, they provide distinct potential targets for novel antibacterial drugs. Vibrio choferae is the most well studied pathogen with a T2S system and, as such, i3 well suited for high throughput screening (HTS) of chemical libraries for small molecule inhibitors of T2S. Chemical inactivation of the T2S system in V. cholerae is likely to result in similar defects as genetic inactivation, blocking secretion of cholera toxin and other potential virulence factors and preventing colonization In the gastro-intestinal tract. Using several unique tools, reagents and assays including a protease secretion assay that is amenable to HTS, we will isolate synthetic compounds that block secretion via the T2S system. The availability of structural, biochemical and in vivo resources will enable us to analyze the mechanism of action of identified Inhibitors. Although the HTS approach does not require prior structural knowledge of the T2S complex, information on the structure and function of this machinery is indispensable for the identification of inhibitor target(s), and for the final development of clinically useful agents.